Phytoremediation of mercury and organomercurials in chloroplast transgenic plants: enhanced root uptake, translocation to shoots, and volatilization

Abstract

Transgenic tobacco plants engineered with bacterial merA and merB genes via the chloroplast genome were investigated to study the uptake, translocation of different forms of mercury (Hg) from roots to shoots, and their volatilization. Untransformed plants, regardless of the form of Hg supplied, reached a saturation point at 200 microM of phenylmercuric acetate (PMA) or HgCl2, accumulating Hg concentrations up to 500 microg g(-1) with significant reduction in growth. In contrast, chloroplast transgenic lines continued to grow well with Hg concentrations in root tissues up to 2000 microg g(-1). Chloroplasttransgenic lines accumulated both the organic and inorganic Hg forms to levels surpassing the concentrations found in the soil. The organic-Hg form was absorbed and translocated more efficiently than the inorganic-Hg form in transgenic lines, whereas no such difference was observed in untransformed plants. Chloroplast-transgenic lines showed about 100-fold increase in the efficiency of Hg accumulation in shoots compared to untransformed plants. This is the first report of such high levels of Hg accumulation in green leaves or tissues. Transgenic plants attained a maximum rate of elemental-Hg volatilization in two days when supplied with PMA and in three days when supplied with inorganic-Hg, attaining complete volatilization within a week. The combined expression of merAB via the chloroplast genome enhanced conversion of Hg2+ into Hg,0 conferred tolerance by rapid volatilization and increased uptake of different forms of mercury, surpassing the concentrations found in the soil. These investigations provide novel insights for improvement of plant tolerance and detoxification of mercury.

FIGURE 1

Hg concentration (µg g⁻¹) in roots and shoots of untransformed (black bars) and chloroplast transgenic lines (pLDR-MerAB and pLDR-MerAB-3-UTR) grown for 15 days in soil amended with 100, 200, and 300 µM of either PMA or HgCl₂. Values shown are the average ± standard deviation of five replicates. (*, Significant difference at P < 0.05 from the untransformed plants of the same treatment. **, Significant difference at P < 0.001 from the untransformed plants of the same treatment. NS, Not significant from the untransformed plants of the same treatment).

FIGURE 2

The relationship between root dry weight (mg/plant) and root Hg concentrations (µg g⁻¹) of untransformed and chloroplast engineered pLDR-merAB and pLDR-merAB 3′UTR tobacco lines. The regression analysis between the dry weight and Hg concentration in tissues is derived from three different treatments with 100, 200, and 300 µM of either PMA or HgCl₂.

FIGURE 3

The relationship between shoot dry weight (mg/plant) and shoot Hg concentrations (µg g⁻¹) of untransformed and chloroplast engineered pLDR-merAB and pLDR-merAB 3′UTR tobacco lines. The regression analysis between the dry weight and Hg concentration in tissues is derived from three different treatments with 100, 200, and 300 µM of either PMA or HgCl₂.

FIGURE 4

Gastight acrylic volatilization chambers used to collect the volatilized mercury from untransformed and chloroplast engineered pLDR-MerAB and pLDR-MerAB 3′UTR tobacco lines (in three replicates) grown over a 13-day period on soil amended with 100 µM of either PMA or HgCl₂. Volatile Hg was quantitatively trapped in alkaline peroxide liquid traps solution (1:1 of 0.1% NaOH and 30% H₂O₂).

FIGURE 5

Rates of Hg[0] volatilization from untransformed (solid line) and chloroplast engineered pLDR-merAB (dotted line) and pLDR-merAB 3′UTR (dashed line) tobacco plants grown for 13 days in soil amended with 100 µM of either PMA (A) or HgCl₂ (B). Values shown are the average of three replicates.